Method for Sequencing RNA by In-source Decay Using Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometer

ABSTRACT

An analytic method is provided for obtaining much sequence information by causing in-source decay of modified RNA and non-modified RNA and generating many fragment ions. Particularly, a method for analysis wherein a matrix that efficiently causes decomposition by in-source decay of RNA of 20 bases or longer is used in an apparatus that has a laser of a wavelength commonly used in MALDI-TOF MS. A specimen containing RNA is subjected to matrix assisted laser desorption ionization time of flight mass spectrometry that uses 2,4-dihydroxyacetophenone as a matrix to obtain fragment ions derived from the RNA. The difference in mass between the peaks of ions in the fragment ions is used to analyze the sequence of the RNA.

TECHNICAL FIELD

The present invention relates to an art for performing RNA sequencing byMALDI-TOF MS. This art is expected to be used in life sciences such asby pharmaceutical companies performing development work on nucleic aciddrugs and companies engaged in the business of synthesis and sale ofoligonucleotides.

BACKGROUND ART

With increased development activities in recent years in nucleic acidpharmaceutical, there is a need for a technology for the analysis ofoligonucleotide sequences of about several dozen bases long. Withnucleic acid drugs, a general practice is to increase in vivo retentionby the introduction of an artificial moiety such as phosphorothioateesters and modified ribose 2′-OH groups. There is a need for atechnology that can sequence nucleic acids that include an artificialmoiety.

Examples of the use of MALDI-TOF MS to sequence oligonucleotides includea method (see Non-Patent Literature 1) wherein a ladder structure massspectrum is obtained for RNA whose phosphodiesters have been partiallyhydrolyzed with an acid and the sequence is analyzed based on the massdifference between peaks, and a method (see Non-Patent Literature 2)wherein a RNA is sequentially decomposed with an endonuclease startingfrom either the 3′-end or the 5′-end, mass spectra are obtained overtime and sequence information is obtained.

In-source decay (ISD) and MALDI-TOF MS are primarily used for theanalysis of amino acid sequence of peptides (see Non-Patent Literature 3and 4). However, there are several reported cases of their use for theanalysis of nucleic acid base sequence (see Non-Patent Literature 5 and6).

With respect to DNA that is 11 bases long, Non-Patent Literature 5discloses the generation of fragment ions by irradiation with laser of awavelength of 266 nm while using picolinic acid as a matrix.

With respect to DNA that is 7 bases long, Non-Patent Literature 6discloses the generation of fragment ions using 2,5-dihydroxybenzoicacid (2,5-DI-113) as a matrix. Even though the literature does notidentify the laser wavelength, based on the use of Voyager Elite(manufactured by Perspective Biosystems), the wavelength is estimated tobe 337 nm.

Non-Patent Literature 7 discloses the analysis of a DNA sequence byin-source decay using a mixture of dihydroxyacetophenone (DHAP) and1,5-diaminonaphtalene (DAN) as a matrix. The literature states thatalmost no fragments were detected when in-source decay was used on RNA.

Non-Patent Literature 8 discloses the use of2,4-dihydroxyacetophenonematrix for the separation of 100-base long DNAand 102-base long DNA consisting of TC repeat sequences on a massspectrum and their detection.

With base sequence analysis of nucleic acids using mass spectrometry,reflecting the cleavage site of the phosphodiester bonds, fragment ionsthat are generated from the nucleic acid are named as a, b, c, or d ifthey possess a 5′-OH group and as w, x, y or z if they possess a 3′-OHgroup (see Non-Patent Literature 9).

However, there are no reports of the analysis of sequences of RNAs(unmodified) and RNAs having modified groups based on the mass spectrumof fragment ions that are generated by in-source decay.

PRIOR ART LITERATURE Non-Patent Literature

-   Non-Patent Literature 1: Bahr U. et al., Anal. Chem., 2009, 81,    3173-3179.-   Non-Patent Literature 2: Gao H. et al., Rapid Commun. Mass    Spectrom., 2009, 23, 3423-3430.-   Non-Patent Literature 3: Takayama M. et al., J. Mass Spectrom. Soc.    Jpn., 2002, 50, 304-310.-   Non-Patent Literature 4: Demeure K. et al., Anal. Chem., 2007, 79,    8679-8685.-   Non-Patent Literature 5: Juhasz P. et al., Anal. Chem., 1996, 68,    941-946.-   Non-Patent Literature 6: Koomen J. M., et al., J. Mass Spectrom.,    2000, 35, 1025-1034.-   Non-Patent Literature 7: Shimadzu Application News, No. B17, 2009    10-   Non-Patent Literature 8: Y. Yoshikawa, K. Nakajima, N. Kimura, M.    Gonda, K. Okamoto, G. Tamiya, H. Inoko, “An efficient application of    MALDI-TOF/MS coupled with microarray for detection of microsatellite    polymorphisms.”, Program Nr: 1239 from 2002 ASHG Annual Meeting,    (online), (search performed on Aug. 18, 2010), Internet <URL:    http://www.ashg.org/geneties/abstracts/abs02/f1239.htm>-   Non-Patent Literature 9: McLuckey, S. A. et al., J. Am. Soc. Mass    Spectrom., 1992, 3, 60-70.

OVERVIEW OF THE INVENTION Problems to Be Solved by the Invention

With the method disclosed in Non-Patent Literature 1, the acidhydrolysis of phosphodiesters of RNA requires a 2′-OH group in theribose. This means that phosphodiesters do not decompose in RNA whose2′-OH group is modified for example by methylation. A problem with thismethod is therefore that the position of the base that is modified bymethylation cannot be identified.

A problem with the method described in Non-Patent Literature 2 is thatit is time-consuming and labor-intensive since various conditions suchas enzymatic digestion time must be considered and mass spectrometry hasto be performed over time.

A problem with the method described in Non-Patent Literature 5 is thatthe wavelength (266 nm) of the laser that is used for the in-sourcedecay analysis of oligonucleotides is uncommon, thus limiting theapparatus that can be used for the analysis to only those apparatuseshaving a laser of the aforesaid wavelength.

Furthermore, with the method according to Non-Patent Literature 5, thereis a description of an example of the analysis of a DNA fragment that is11 bases long, but there is no description of the analysis usingin-source decay of RNA of 20 bases or longer which are the objects ofstudies in the context of nucleic acid pharmaceutical.

The DNA that is analyzed in Non-Patent Literature 6 is seven bases longand the fragment ions that are obtained by in-source decay are few innumber, thus providing only a partial sequence information and creatinga problem that the sequence of RNA of 20 bases or longer which are beingstudied in the context of nucleic acid pharmaceutical cannot beanalyzed.

As the literature states, a problem with the method according toNon-Patent Literature 7 is that the sequence of RNA cannot be analyzedby the in-source decay.

With Non-Patent Literature 8, what are being detected are solely theparent ions of a long-chain DNA. Fragment ions that allow sequencing arenot detected.

The decomposition mechanism of peptides by in-source decay is believedto be triggered by the addition of a hydrogen atom to a carbonyl group.The cleavage of oligonucleotides by in-source decay is believed to becaused by the addition of hydrogen atom to a phosphodiester bond. Thisdecomposition is expected to occur regardless of whether 2′-OH ispresent.

Non-Patent Literature 5 discloses that, according to the in-source decayfragment efficiency of oligonucleotides, the ion strength that can beobtained will be only several % of intact ions. Hence, to obtainin-source decay ions in abundance, a matrix is required whose ionizationefficiency of the oligonucleotide is high.

In light of the above, it is the object of the present invention toprovide an analytical method that can provide much sequence informationby causing in-source decay of non-modified RNA and modified RNA andgenerating many fragment ions.

It is also the object of the present invention to provide an analyticalmethod that uses a matrix that can efficiently cause decomposition byin-source decay of RNA whose length is 20 bases or longer using anapparatus having a laser of a wavelength commonly used with MALDI-TOFMS.

Solution

After diligent work, the present inventor discovered that2,4-dihydroxyacetophenone efficiently causes in-source decay of RNA. Thepresent inventor also discovered that commonly used nitrogen laser witha wavelength of 337 nm generated fragment ions by in-source decay whenused with 2,4-dihydroxyacetophenone. The present inventor alsodiscovered that 2,4-dihydroxyacetophenone causes in-source decay of RNAthat includes modifications.

The present inventor completed the present invention based on the aboveknowledge.

The present invention includes the following inventions.

(1) A method for sequencing of RNA wherein a specimen including RNA issubjected to matrix assisted laser desorption ionization time of flightmass spectrometry that uses 2,4-dihydroxyacetophenone as a matrix toobtain fragment ions derived from the RNA by in-source decay and thesequence of the RNA is analyzed by the difference in mass between peaksof the fragment ions.

In the above, the RNA includes both non-modified RNA sand modified RNAs.

(2) The method for sequencing RNA described in (1) wherein the RNA hasribose with a 2′ modified group.

(3) The method for sequencing RNA described in (2) wherein the RNA has aribose with a 2′-O-methyl group.

(4) The method for sequencing RNA described in any one of either (1)through (3) wherein the RNA has a base length of 20 to 30.

Effects of the Invention

The present invention provides an analytic method wherein in-sourcedecay of non-modified RNA and modified RNA is used to generate manyfragment ions and thus to obtain much sequence information.

The present invention provides an analytic method wherein an apparatushaving a laser of a commonly used wavelength with MALDI-TOF MS is usedwith a matrix that efficiently causes decomposition by in-source decayof RNA.

With the present invention, at least 90%—for example—of ions of anentire sequence can be assigned by the assignment of fragment ions thatare characteristic of w-series, y-series and d-series fragment ions thatare generated by in-source decay of the RNA being analyzed.

The present invention provides an analytic method that is useful inanalyzing the sequence of RNA of 20 mers or longer which is the subjectof nucleic acid pharmaceutical. The present invention also provides asimple means for the analysis of oligonucleotide sequences that does notrequire a pre-process such as an acid treatment.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the estimated mechanism by which RNA decomposes into aw-series by in-source decay. In the equation, B₁ and B₂ represent bases.

FIG. 2 shows a mass spectrum of fragment ions that were generated byin-source decay of RNA that is 21 bases long whose adenosine at theeighth base from the 5′ end is 2′-O-methylated (5′-UCG AAG U(Am)U UCCGCG UAC GdTdT-3′ where Am represents 2′-O-methyladenosine). The spectrathat are shown used: 2,4-DHAP; 2,5-DHAP (for comparison); 2,6-DHAP (forcomparison) and 2,4,6-THAP (for comparison) as the matrix.

FIG. 3( a) shows the mass spectrum of fragment ions that were generatedby the in-source decay of non-methylated RNA that is 21 bases long. FIG.3( b) shows the mass spectrum of fragment ions that were generated byin-source decay of RNA whose adenosine at the eighth base from the 5′end is 2′-O-methylated.

FIG. 4( a) shows the mass spectrum of fragment ions that were generatedby the in-source decay of 2′-O-methylated RNA. For comparison, FIG. 4(b) shows the mass spectrum of 2′-O-methylated RNA that was pre-treatedwith an acid treatment.

FIG. 5 shows the naming convention for RNA fragment ions. The structuralequation of the RNA has been simplified for all parts except for thephosphate bond sites. n₁, n₂, and n₃ represent a nucleoside moiety.

EMBODIMENTS 1. RNA

No particular limitations are imposed on the RNA (ribonucleic acid) thatis covered by the present invention as long as the RNA has a base moietyof adenine (A), guanine (G), cytosine (C) and uracil (U), a sugar moietyof a ribose and a phosphate moiety of phosphate bonds. Also, with thepresent invention, the RNA includes both natural RNA and artificial RNAanalogs. (In the specification, the term “non-modified RNA” may be usedto refer to a type of natural RNA and the term “modified RNA” may beused to refer to a type of an artificial RNA analog.)

No particular limitations are imposed on the type of an artificial RNAanalog. It may be a RNA whose base moiety is modified, whose sugarmoiety is modified or whose phosphate moiety is modified. However, it ispreferable for the modification to be acceptable in the field of nucleicacid pharmaceutical.

An example of a RNA whose base moiety that is modified is methylatedcytosine.

Examples of RNA whose sugar moiety is modified are an RNA whose ribosehas a 2′ modification group, and linked nucleic acid (LNA) whose 2′position is bonded to the 4′ position. Preferable examples include RNAwhose ribose has a 2′-0-methyl group (2′-O-methylated RNA) and RNA whoseribose has a 2′-F group (2′-fluorinated RNA).

An example of RNA whose phosphate moiety is modified is phosphorothioateRNA wherein an oxygen atom in a phosphodiester bond (P═O) is replaced bya sulfur atom.

No limitations are imposed on the base length of the RNA, but a smallRNA is preferred. One possibility is length of up to 30 bases long(e.g., 20 to 30 bases long). If the RNA is for nucleic acidpharmaceutical, 20 to 25 bases long is preferable, and 21 to 23 baseslong is more preferable. If the RNA is a medical metabolite or if it isnot limited to just pharmaceutical, the length can be less than 20 baseslong.

No limitations are imposed on the amount of RNA that is used in thepresent invention. In particular, because the present invention isuseful in handling very small quantities of RNA, the quantity of the RNAmay be in the picomole level, e.g., 5 to 20 picomoles.

2. Matrix

With the present invention, 2,4-dihydroxyacetophenone is used as thematrix.

Fragment ions can be generated with widely used laser of the wavelengthof 337 nm by using 2,4-dihydroxyacetophenone, and decomposition byin-source decay is efficient. No limitations are imposed on the amountof 2,4-dihydroxyacetophenone that is used, and those skilled in the artcan decide the amount of 2,4-dihydroxyacetophenone to use just like anyother matrix. For example, the quantity may be 2,000 to 50.000-fold (ona molar basis) of the RNA that is analyzed.

2,4-dihydroxyacetophenone is dissolved in a suitable solution and used.No limitations are imposed on the composition of the solution, and thoseskilled in the art may select a solution as deemed fit. For example,2,4-dihydroxyacetophenone may be used as an aqueous solution of anorganic solvent such as acetonitrile or methanol. No limitations areimposed on the concentration of the organic solvent, but an example is30% to 50% (by volume).

With the present invention, it is preferable to use2,4-dihydroxyacetophenone as the only matrix and not to mix with someother matrix. In particular, 2,4-dihydroxyacetophenone is not used as amixture with 1,5-diaminonaphtalene (DAN) which is known as a matrix thatefficiently causes in-source decay.

Matrix additives may be used with the present invention. Ammonium saltsof an organic acid or inorganic acid may be used as a matrix additive.Specific examples include ammonium citrate dibase (ACDB), ammoniumacetate (AA), ammonium chloride (ACl), ammonium citrate tribase (ACTB),ammonium fluoride (AF) and ammonium tartarate (AT).

No limitations are imposed on the amount of additives that are used, andthe amount can be suitably decided by those skilled in the art. Anexample would be one-fourth to an equal amount (on a molar basis) as2,4-dihydroxyacetophenone which is used as the matrix.

3. Mass Spectrometry

The RNA is mixed with the matrix and subjected to mass spectrometry.With the mass spectrometry, matrix assisted laser desorption ionizationtime of flight (MALDI-TOF) mass spectrometer is used, and fragment ionsderived from the RNA are obtained by in-source decay (ISD). WithMALDI-TOF mass spectrometry that uses in-source decay, the RNA isirradiated with a laser to simultaneously fragment the RNA inside theion source. Excited molecular ions, i.e., fragment ions that aregenerated by the fragmentation, are detected to obtain RNA sequenceinformation. Because fragment ions generated by in-source decay aredetected in both the positive and negative modes, no limitations areimposed on the detection mode with regards to polarity, but the negativemode which is generally used for the measurement of oligonucleotides ispreferred.

FIG. 1 shows the estimated in-source decay mechanism of RNA. As FIG. 1shows, it is believed that the decomposition of RNA by in-source decayis triggered by the addition of hydrogen to the oxygen atom (P═O) inphosphodiester bond.

Similarly, the decomposition of a phosphorothioate type RNA by in-sourcedecay is believed to be triggered by the addition of a hydrogen atom tothe sulfur atom (P═S).

This means that decomposition by in-source decay occurs regardless ofwhether the modification is to the base moiety, the sugar moiety (2′hydroxyl group) or the phosphate moiety of the RNA.

With a method for RNA sequencing according to the present invention, byusing the afore-described matrix, the in-source decay of RNA isefficiently performed, and fragment ions that are sufficient forproviding the sequence information are obtained as w-series, y-seriesand d-series ions. Names are assigned to the respective fragment ions ofthe RNA based on the oligonucleotide naming convention shown in FIG. 5.The w-series fragment ions represent a series that are not generated byacid treatment and the like and are characteristic of in-source decay.The mass spectra that are obtained with an in-source decay represent themass of the nucleotides that constitute the RNA as represented by thedifference in mass between peaks of the ions in each series. Hence, byreading the difference in mass between the peaks, RNA can be very easilysequenced.

EMBODIMENTS

The present invention is further described next in detail with referenceto its embodiments. It should be noted that the present invention is notlimited to the embodiments described here. Unless specifically statedotherwise, all quantities represented in percent (%) are based onvolume. The mass spectra that are shown for the embodiments plot themass/charge ratio (m/z) along the horizontal axis and relative intensityalong the vertical axis.

Embodiment 1

RNA samples were subjected to in-source decay analysis using as thematrix the following four compounds whose structure is shown below:2,4-dihydroxyacetophenone; 2,5-dihydroxyacetophenone (for comparison);2,6-dihydroxyacetophenone (for comparison) and2,4,6-trihydroxyacetophenone (for comparison). The RNA samples that wereanalyzed were 21 bases long with a 2′-O-methylated adenosine positionedas the eighth base from the 5′ end. The specific sequence is 5′-UCG AAGU(mA)U UCC GCG UAC GdTdT-3′. (UCG AAG U(mA)U UCC GCG UAC G is identifiedas sequence number 1, and “mA” represents 2′-O-methyladenosine.) ThisRNA sample is identified hereafter as 2′-O-methylated RNA.

Each matrix was dissolved in an aqueous solution that included 70 mM ofammonium citrate dibase and 50% acetonitrile. The 2,4-DHAP, 2,6-DHAP and2,4,6-THAP were formulated to a concentration of 20 mg/ml in the aqueoussolution while the 2,5-DHAP was formulated to a concentration of 10mg/ml to prepare the matrix solution.

The 21-base long, 2′-O-methylated RNA with a concentration of 50 pmol/μlwas mixed at a ratio of 1:1 (volume ratio) with a matrix solution. Themixed solution was applied to a stainless steel plate for MALDI-TOF MSmeasurement use, allowed to dry and subjected to MALDI-TOF MSmeasurement. AXIMA Confidence (registered trademark) manufactured byShimadzu Corporation operating in the linear and negative mode was usedfor the MALDI-TOF MS measurement.

FIG. 2 shows the mass spectrum that was obtained by the MALDI-TOF MSmeasurement. The ion intensity of the molecular related ion (m/z of6658) of the RNA that was analyzed was the strongest when using 2,4-DHAPas the matrix as compared to the use of other matrices. Also as shown inFIG. 2, fragment ions in the vicinity of between m/z of 3500 and m/z of6000 were detected with the strongest intensity as compared to the othermatrices, indicating the high in-source decay efficiency.

Embodiment 2

Using 2,4-DHAP as the matrix, MALDI-TOF MS measurements were performedon 2′-O-methylated RNA and RNA that was not 2′-O-methylated(non-methylated RNA) in the same manner as with Embodiment 1. Thespecific sequence of the non-methylated RNA was 5′-UCG AAG UAU UCC GCGUAC GdTdT-3′. (UCG AAG UAU UCC GCG UAC G is identified as sequencenumber 2.)

FIG. 3 shows the mass spectrum of the fragment ions that were generatedby in-source decay ((a): non-methylated RNA and (b): 2′-O-methylatedRNA). w-series fragment ions are detected, and, among the 21 bases, allbases except for 2 bases at the 3′ end could be associated with theirions, thus sequencing the RNA. The position of the methylation-modifiedbase (mA) could also be confirmed.

Comparison Example 1

2′-O-methylated RNA samples were prepared as 10 pmol/μl aqueoussolution. A reagent solution was prepared containing 3-hydroxypicolinicacid (3-HPA) at a concentration of 50 mg/ml in an aqueous solution of 5%trifluoroacetic acid.

The afore-described sample aqueous solution and the afore-describedreagent solution were mixed in the same volume to prepare a reactionmixture solution (i.e., trifluoroacetic acid with a final concentrationof 2.5%). 1 μl of the reaction mixture solution was immediately appliedto a stainless steel plate for MALDI use and air-dried. 0.5 μl of 10mg/ml ammonium citrate dibase aqueous solution was applied to the samedried spot and further air dried. After drying, measurements were takenusing MALDI-TOF MS

FIG. 4( b) shows the mass spectra obtained by MALDI-TOF MS measurementafter an acid treatment. FIG. 4( a) shows an enlarged view of a portion(m/z between 3700 and 4600) of the mass spectra shown in FIG. 3( b) fora 2′-O-methylated RNA sample obtained as embodiment 2.

Because, as FIG. 4( b) shows, y13 was not detected after the acidtreatment, it was confirmed that methylation site was not severed. Onthe other hand, as FIG. 4( a) shows, since the w13 ion was detected inthe fragment ions generated by in-source decay, it was confirmed thatthe phosphodiester bond was broken regardless of methylation or not.

Sequence Listing Free Text

Sequence number 1 is a synthetic oligonucleotide whose eighth positionis 2′-O-methyladenosine.

Sequence number 2 is a synthetic oligonucleotide.

1. A method for sequencing RNA, comprising: subjecting a specimencontaining RNA to matrix assisted laser desorption ionization time offlight mass spectroscopy using 2,4-dihydroxyacetophenone as a matrix toobtain fragment ions derived from said RNA by in-source decay; andanalyzing said RNA sequence by the difference in mass between peaks ofsaid fragment ions.
 2. The method for RNA sequencing according to claim1 wherein said RNA has a 2′ modified group in the ribose.
 3. The methodfor RNA sequencing according to claim 2 wherein said RNA has a2′-O-methyl group in the ribose.
 4. The method for RNA sequencingaccording to claim 1, wherein said RNA has a length of between 20 and 30bases.